Prodrugs of DP IV-inhibitors

ABSTRACT

The present invention relates to prodrug compounds of inhibitors of dipeptidyl peptidase IV (DP IV), which prodrug compounds have the general formula A-B-C, wherein  
     A is an amino acid,  
     B is a chemical bond between A and C or is an amino acid, and  
     C is a stable inhibitor of DP IV.  
     Such prodrug compounds are used in the treatment of impaired glucose tolerance, glucosuria, hyperlipidaemia, metabolic acidoses, diabetes mellitus, diabetic neuropathy and nephropathy and of sequelae of diabetes mellitus in mammals.

[0001] According to the invention there are provided prodrug compoundsof inhibitors of dipeptidyl peptidase (DP IV), which prodrug compoundshave the general formula A-B-C, wherein

[0002] A is an amino acid,

[0003] B is a chemical bond between A and C or is an amino acid, and

[0004] C is a stable inhibitor of DP IV.

[0005] It has been found that administering inhibitors (effectors) of DPIV or DP IV-analogous enzyme activity in the blood of a mammal causes,because of the associated temporary reduction in enzyme activity,reduced breakdown of the endogenous (and also exogenously administered)insulino-tropic peptides Gastric Inhibitory Polypeptides 1-42 (GIP₁₋₄₂)and Glucagon-Like Peptide Amides-1 7-36 (GLP-1₇₋₃₆) (or also GLP-1₇₋₃₇or analogues thereof) by DP IV and DP IV-like enzymes and, accordingly,the fall in concentration of those peptide hormones, or analoguesthereof, is reduced or delayed. The greater stability of (endogenouslypresent or exogenously introduced) incretins or analogues thereof, whichresults from the action of DP IV-effectors, increases their availabilityfor insulinotropic stimulation of the incretin receptors of theLangerhans cells in the pancreas and alters, inter alia, theeffectiveness of the body's own insulin, resulting in stimulation ofcarbohydrate metabolism in the treated organism. As a result, in theserum of the treated organism, the blood sugar level drops below theglucose concentration that is characteristic of hyperglycaemia.Consequently, by means of DP IV-inhibitors it is possible to prevent orto mitigate metabolic anomalies such as excess weight, glucosuria,hyperlipidaemia and also possible serious metabolic acidoses anddiabetes mellitus which are a consequence of prolonged elevated glucoseconcentrations in the blood [see DE 196 16 486].

[0006] With the aid of DP IV-inhibitors, it is also possible,experimentally, to prevent the penetration of CD 26 (DP IV) positivecells by HIV [see WAKSELMAN, M., NGUYEN, C., MAZALEYRAT, J.-P.,CALLEBAUT, C., KRUST, B., HOVANESSIAN., A. G., Inhibition of HIV-1infection of CD 26+ but not CD 26− cells by a potent cyclopeptidicinhibitor of the DPP IV activity of CD 26. Abstract P 44 of the 24^(th)European Peptide Symposium 1996].

[0007] It has also been found that DP IV can modulate the activity ofneuroactive peptides, such as neuropeptide Y and CLIP [see MENTLEIN, R.,DAHMS, P., GRANDT, D., KRUGER, R., Proteolytic processing ofneuropeptide Y and peptide YY by dipeptidyl peptidase IV. Regul. Pept.49, 133 (1993); WETZEL, W., WAGNER, T., VOGEL, D., DEMUTH, H.-U.,BALSCHUN, D., Effects of the CLIP fragment ACTH 20-24 on the duration ofREM sleep episodes. Neuropeptides, 31, 41 (1997)].

[0008] The problem of the present invention is to provide effectors ofDP IV which have an increased action compared with known inhibitors andwhich have a temporally defined onset of action.

[0009] The problem is solved by providing prodrug compounds ofinhibitors of dipeptidyl peptidase IV (DP IV), which prodrug compoundshave the general formula A-B-C, wherein

[0010] A is an amino acid,

[0011] B is a chemical bond between A and C or is an amino acid, and

[0012] C is a stable inhibitor of DP IV.

[0013] Surprisingly, inhibitors of that kind which are masked asprodrugs have significantly increased activity compared with non-maskedinhibitors: When identical amounts of non-masked DP IV-inhibitors and ofprodrug compounds according to the invention are used, an improvement inglucose tolerance of up to 75% is obtained in Wistar rats; see alsoTable 4.

[0014] That improvement is all the more astonishing in view of the factthat it has been found that 100% of non-masked inhibitors of DP IV areabsorbed from the gastrointestinal tract of mammals and enter thevascular compartment of the body. It might, therefore, have beenexpected that prodrug compounds, which normally are intended only toprevent degradation of orally administered compounds in thegastrointestinal tract, would not lead to an increase in the activity ofthe inhibitors. Furthermore, it should be mentioned just in passing thatthere was no cause whatever for a person skilled in the art, on thebasis of those facts, to look for modified inhibitors, even thoughprodrug compounds were known per se; see e.g. PCT/US 97/09421.

[0015] According to a preferred embodiment of the present invention,prodrug compounds are used in which B is proline, hydroxyproline,thiazolidinecarboxylic acid, dehydroproline, pipecolic acid,azetidinecarboxylic acid or aziridine-carboxylic acid, with proline andhydroxyproline being especially preferred. B preferably represents apeptide bond between A and C or is linked to A and C via peptide bonds.

[0016] The compounds according to the invention also have the advantageespecially that the inhibitors of DP IV are released according toindividual patient needs:

[0017] When a prodrug compound according to the invention interacts witha DP IV molecule, it is cleaved by the enzyme into the groups A-B andthe inhibitor C. The inhibitor C will inhibit the DP IV molecule so thatthe latter cannot cleave any further prodrug compounds. When further DPIV molecules are present, prodrug compounds will be cleaved (if asufficient amount of prodrug compounds has been administered) until thelast DP IV molecule has been inhibited. The remaining prodrug compoundsare not degraded and thus constitute an inhibitor reservoir until theconcentration of DP IV molecules increases again or inhibitor moleculesare displaced by DP IV or inhibitor molecules are eliminated, and theprodrug compounds are cleaved again, thus releasing inhibitors.

[0018] The invention therefore has the further advantage that eachorganism will release precisely the amount of inhibitor that isnecessary to inhibit DP IV, which is present in different amounts inindividual cases. If, for example, a patient has a high concentration ofDP IV, a large amount of inhibitor will be released; if there is only aslightly elevated concentration of DP IV, only a small amount ofinhibitor will be released.

[0019] Furthermore, preference is given, according to the invention, toprodrug compounds wherein C is an amino-acylpyrrolidide,aminoacylthiazolidide or N-dipeptidyl, O-acyl hydroxylamine. Suchinhibitors have shown themselves to be especially active DPIV-inhibitors. As examples of such inhibitors there may be mentioned,for example, Ile-Thia, Ile-Pyr, Val-Thia and Val-Pyr.

[0020] The inhibitors (component C) can, according to the invention,also be present in salt form, with organic salts such as acetates,succinates, tartrates or fumarates or inorganic acid radicals such asphosphates or sulphates being preferred. Fumarates are especiallypreferred.

[0021] Special preference is given to compounds wherein A-B is adipeptide of formula IIe-Pro or Gly-Pro.

[0022] The present invention therefore relates to novel prodrugcompounds of inhibitors of the serine peptidase dipeptidyl peptidase IV,which prodrug compounds can be used in the treatment of variousdisorders, especially metabolic disorders associated with diabetesmellitus.

[0023] A further advantage of the prodrug compounds according to theinvention lies in the fact that it is possible for the onset of actionand also the duration of action of the DP IV-inhibitors to be temporallycontrolled by suitable selection of the groups A-B. In particular, therelease of the groups A-B from the prodrug compounds according to theinvention depends upon the nature of the amino acid radical of A: Inrespect of the definition of group A, the following sequence has beenfound, in particular, for the rate at which the radicals A-B arereleased from the prodrug compounds A-B-C by DP IV:Ile<Val<Phe<Pro<Ala<Gly. The rate constants of the corresponding DPIV-catalysed release reactions are between 1 s⁻¹ and 100 s⁻¹. A means isthus available for releasing the DP IV-inhibitors in a preciselytemporally defined manner: When the enzymes are to have an immediateonset of action, for example upon ingestion of glucose-rich nutrient, acompound A-B-C will be selected that has, for example, the amino acidGly as group A; when the onset of action of the inhibitor is to bedelayed, the amino acid Ile, for example, can be selected as group A. Bymeans of the prodrug compounds according to the invention, therefore, itis possible for DP IV-inhibitors to be transported through the mucosa ofthe small intestine especially almost without a delay, for examplealmost simultaneously with ingested nutrients.

[0024] When B represents a bond, it is especially a peptide bond; when Brepresents an amino acid, it is linked to A and C preferably via peptidebonds.

[0025] On analysis of the dose-effect relationships of the DPIV-inhibitor isoleucyl thiazolidide as a modulator of the blood glucoseconcentration in the mammalian organism, a difference can be foundbetween oral and parenteral administration of the active substance toWistar rats: On oral administration, saturation was observed in theuptake of the active substance (measured on the basis of inhibition ofthe serum enzyme), whereas on parenteral administration of the inhibitorcomplete inhibition of the enzyme was observed. That is demonstrated, byway of example, in Table 1: TABLE 1 Residual activity of DP IV withrespect to 0.4 mM of the substrate H-Gly-Pro-pNA at 30° C., pH 7.6 andan ionic strength of 0.125, after i.v. and p.o. administration and as afunction of the isoleucyl thiazolidide (Ile-Thia) dose, determined 30min after administration of the inhibitor. Ile-Thia dose Ile-Thia doseon parenteral DP IV activity on oral DP IV activity administration in %administration in %   0 mg 100   0 mg 100 0.02 mg 80 2.5 mg 52  0.2 mg32 5.0 mg 40   2 mg 5  10 mg 28   20 mg 0  20 mg 29

[0026] In view of the fact that in the intestine there are also presentenzymes, especially high concentrations of DP IV, that are capable ofcleaving the cleavable groups of prodrugs and consequently of releasingthe medicament and also—as already mentioned—it has been found that DPIV-inhibitors are absorbed quantitatively from the gastro-intestinaltract, it was to be expected that the use of prodrug compounds of DPIV-inhibitors would not bring about any improvement in that situation.

[0027] It was, therefore, extremely surprising to find that the prodrugsof DP IV-inhibitors according to the invention bring about clearlyenhanced glucose tolerance in the glucose tolerance test compared withthe corresponding non-masked DP IV-inhibitors. That behaviour wasespecially surprising because—as mentioned hereinbefore—it is possiblefor the prodrugs to be cleaved already in the intestine by enzymespresent therein such as dipeptidyl peptidase and therefore, exactly likethe non-masked inhibitors, ought no longer to be available for transportto the target site:

[0028] As soon as the prodrug compounds are cleaved by DP IV or otherenzymes present in the intestine, the inhibitors according to theinvention are released, which causes inhibition of DP IV in exactly thesame manner as when non-masked inhibitors are used. Consequently, nofurther breakdown of the prodrug compounds by DP IV takes place; allprodrug compounds that are still undegraded or additionally introduced,as well as excess (that is to say, not bound to DP IV) non-maskedinhibitors, can pass undegraded from the gastrointestinal tract into thevascular compartment of a body. There they can then be used as DPIV-inhibitors according to individual needs, as mentioned hereinbefore.However, after a certain time, the inhibitors bound to DP IV in theintestine are released again and enter the vascular compartment.

[0029] With the aid of the prodrug compounds according to the invention,it is therefore also possible to obtain a desired increase in action invivo.

[0030] Moreover, it is possible to control the site of release andaction of the DP IV-inhibitors by means of the nature of the radicalsA-B:

[0031] Various other aminopeptidases such as, for example, pyroglutamylaminopeptidase and prolyl aminopeptidase are present in the blood ofmammals, in addition to dipeptidyl peptidase IV. By suitable selectionof the radicals A-B, it is possible according to the invention topredetermine which aminopeptidase is to release the DP inhibitor and soto determine where the action of the inhibitor is to occur. The prodrugcompounds according to the invention or corresponding pharmaceuticalcompositions can therefore be used in cell-, tissue- or organ-specificinhibition of DP IV. It is also possible to select the groups A-B sothat only those enzymes that are present in the vascular compartment andthat release the inhibitors at a sufficiently fast rate are targeted.

[0032] To summarise, it may be stated that, by means of the prodrugcompounds of DP IV-inhibitors according to the invention, it ispossible, in an entirely surprising manner:

[0033] 1. to achieve increased action of the inhibitors;

[0034] 2. for the inhibitors to be released according to patient needs;

[0035] 3. for the inhibitors to be released from the prodrug compoundsin a temporally controlled manner;

[0036] 4. for the site at which the inhibitors are released from theprodrug compounds to be controlled; and

[0037] 5. for a reservoir of DP IV-inhibitors to be provided.

[0038] According to the invention, pharmaceutical compositions,especially for oral administration, are also provided, which arecharacterised in that they comprise at least one prodrug compoundaccording to the invention optionally in combination with customarycarriers or excipients.

[0039] The prodrug compounds or pharmaceutical compositions comprisingthem in accordance with the invention can be used in the treatment orprophylaxis of disorders in mammals that can be treated by modulatingthe DP IV activity of a mammal, such as, for example, metabolicdisorders in humans.

[0040] In particular, they can be used in the treatment of impairedglucose tolerance, glucosuria, hyperlipidaemia, metabolic acidoses,diabetes mellitus, diabetic neuropathy and nephropathy and of sequelaeof diabetes mellitus in mammals.

EXAMPLES

[0041] 1. Synthesis of Prodrug Compounds According to the Invention

[0042] 1.1. Synthesis of H-Pro-Ile-Thia/HCl

[0043] 6.5 mM Boc-Pro-Ile-OH (one equivalent=1 eq.) is suspended,together with N-hydroxybenzotriazole (1 eq.) and thiazolidine (1 eq.),in 30 ml of dichloromethane (DCM). The equivalent amount of 1Mdicyclohexylcarbodiimide solution is added dropwise, at −10° C., withstirring. Stirring is carried out at −10° C. and overnight at roomtemperature. For the purpose of working-up, the solution is thoroughlyfiltered off from the dicyclohexylurea that is precipitated out, DCM isdrawn off in vacuo and the residue obtained is taken up in ethylacetate. The ethyl acetate solution is washed at least three times withsaturated bicarbonate solution, once with saturated NaCl solution, threetimes with dilute KHSO₄ solution and again with NaCl solution. The ethylacetate phase is dried over Na₂SO₄ and concentrated using a rotaryevaporator, and the remaining crude product is re-crystallised usingethyl acetate/pentane. Boc-Pro-Ile-Thia crystallises after 1-2 days at4° C. (yield 80%). 1.1N HCl/glacial acetic acid solution is added toBoc-Pro-Ile-Thia (3 ml per mmol of peptide). Stirring is carried out fortwo hours at RT, absolute ether is added and excess removal solution isevaporated off using a rotary evaporator. The hydrochloride crystallisesquantitatively under absolute ether overnight at 4° C. The crystals arequickly separated off by suction filtration, washed several times withabsolute ether, and the product is stored in a desiccator over KOH orphosphorus pentoxide.

[0044] 1.2. Synthesis of H-Gly-Pro-Ile-Thia/HCl

[0045] Boc-Gly-OH (1 eq.) is dissolved in 20 ml of tetrahydrofuran(THF), cooled to −10° C. and, with stirring, N-methyl-morpholine (1 eq.)and chloroformic acid isobutyl ester (1 eq.) are added in succession.Activation is carried out for about 20 min. In parallel,Pro-Ile-Thia.HCl (1 eq.) is suspended in 10 ml of THF, equilibrated to−10° C., and N-methylmorpholine (1 eq.) is added for the purpose ofneutralisation. After completion of the activation time, both solutionsare mixed together and, after one to two hours, heated to roomtemperature and stirred overnight. A small amount of water is then addedto the reaction mixture and the THF is distilled off in vacuo. Theremaining residue is taken up in ethyl acetate and washed at least threetimes with saturated sodium bicarbonate solution, once with saturatedNaCl solution, three times with dilute KHSO₄ and again with saturatedNaCl solution. The ethyl acetate phase is dried over Na₂SO₄,concentrated using a rotary evaporator and the productBoc-Gly-Pro-Ile-Thiazolidide is re-crystallised using ethylacetate/pentane (yield 85%). The removal of Boc is carried outanalogously to the synthesis of H-Pro-Ile-Thia/HCl (yield >95%). TABLE 2Analytical data for prodrugs of inhibitors of dipeptidyl peptidase IVMW, CE purity, HPLC Melting calculated* MW, found retention purity pointSubstance [g/mol] M + H⁺ time (Rt) Rt ° C. pGlu-Ile-Thia*HCl 349.84314.8 4.2 min  10.4 min 30-40 Pro-Ile-Thia*HCl 335.90 300.8 4.5 min10.05 min 45-69 Gly-Pro-Ile-Thia*HCl 392.94 357.8 4.6 min  8.8 min111-121 Ile-Pro-Ile-Thia*HCl 449.05 413.6 5.6 min  10.0 min  98-107Pro-Pro-Ile-Thia*HCl 433.01 397.6 5.3 min 11.35 min 101-118 Conditionsfor the analysis: HPLC Column: LiChrospher 250-4, 100 RP-18.5 μm,temperature 25° C. Eluant: 30% ACN, 0.1% TFA, isocratic, flow rate 0.5ml/min Detection wavelength: 210 nm CE Capillary: 30 cm × 50 μm fusedsilica, temperature 25° C. Detection wavelength: 200 nm Injection: 5sec, 50 mbar Separation: 0.1 M Na phosphate buffer, pH 2.5; duration 7min at 12 kV

[0046] 2. Affinity and Transport of Various Peptides, DP IV-Inhibitorsand Prodrugs to the Peptide Transporter PepT1

[0047] The affinity of various peptides, DP IV-inhibitors and prodrugsof inhibitors of DP IV to the peptide transporter PepT1 were analysed bydisplacement of the radioactively labelled substrate D-Phe-Ala (AMASHEH,S., WENZEL, U., WEBER, W. M., CLAUSS, W., DANIEL, H.,Electrophysiological analysis of the function of the mammalian renalpeptide transporter expressed in Xenopus laevis oocytes. J. Physiol.504, 169-174 (1997)]. It is shown that, for example, the tetrapeptidederivative Ile-Pro-Ile-Thia becomes bound to the transporter proteinPepT1 in a comparable manner to, or better than, selected amino acidderivatives and, in comparison with selected amino acid and peptideanalogues, is transported in a similar or better manner (Table 3). TABLE3 Transport properties of various amino acid and peptide derivatives onthe human peptide transporter PepT1 Electrophys. transport Amino acid oranalysis (hPEPT1 expr. in Binding peptide oocytes), flux % based onconstant, mM, to PepT1, derivative the Gly-Gln control (100%) relativeto D-Phe-Ala Lys-phe 95 0.08 Lys-Phe-Pro 10 0.19 Asn-Pyr 30 3.01Asn-Thia 83 0.50 His-Pyr 7 5.34 His-Thia 12 0.57 Ile-Pyr 14 2.66Ile-Thia 25 0.98 Ile-Pro-Ile-Thia 44 0.61

[0048] Release of the active DP IV-inhibitor Ile-Thia from prodrugsaccording to the invention in human whole blood

[0049] According to an embodiment of prodrugs of DP IV-inhibitorsaccording to the invention, retarded release of DP IV-inhibitors in thetarget compartment, for example, in the blood circulation, is alsopossible.

[0050]FIG. 1 shows, by way of example, the inhibition of human blood-DPIV resulting from release of the inhibitor isoleucyl thiazolidide fromprodrug compounds according to the invention, the inhibition followingdifferent courses as a function of time. Release of the masked DPIV-inhibitor in the blood can be carried out, in the case of theexamples selected, by DP IV itself (Pro-Pro-Ile-Thia=PPIThia,Gly-Pro-Ile-Thia=GPIThia) or by aminopeptidases (pGlu-Ile-Thia=pEIThia,Pro-Ile-Thia ═PIThia) (FIG. 1). When the same concentration of prodrugcompound is used, there is a difference in the efficiency with which theDP IV-inhibitor isoleucyl thiazolidide is released from the prodrugcompounds in the blood, a more marked delay in the release of activesubstance being shown in the case of Pro-Pro-Ile-Thia (PPI Thia) andpGlu-Ile-Thia (pEI Thia) compared with Pro-Ile-Thia (PI Thia) andGly-Pro-Ile-Thia (GPI thia).

[0051] 3. Enhancement of DP IV-Inhibitor-Imparted Glucose ToleranceResulting From the use of Prodrugs

[0052] As a result of converting the active substance isoleucylthiazolidide into prodrugs according to the invention, a markedlyimproved profile of action is observed in the Wistar rat following oraladministration (FIG. 2). The desired reduction in the level of bloodglucose caused by DP IV-inhibitors in the time period examined isenhanced by about 30% by using the prodrug compounds according to theinvention as opposed to the non-masked active substance Ile-Thia (Table4): TABLE 4 Relationship of blood glucose level within 100 minutes ofp.o. glucose stimulation and p.o. administration of Ile-Thia or prodrugsaccording to the invention to Wistar rats (dose: 2.5 μM activesubstance/300 g of animal) Active substance/prodrug % glucose levelcontrol 100 Ile-Thia 74.4 Gly-Pro-Ile-Thia 57.1 Pro-Ile-Thia 56.1

1. Prodrug compounds of inhibitors of dipeptidyl peptidase IV (DP IV),which prodrug compounds have the general formula A-B-C, wherein A is anamino acid, B is a chemical bond between A and C or is an amino acid,and C is a stable inhibitor of DP IV.
 2. Prodrug compounds according toclaim 1, characterised in that B is proline, hydroxyproline,thiazolidinecarboxylic acid, dehydroproline, pipecolic acid,azetidinecarboxylic acid or aziridinecarboxylic acid.
 3. Prodrugcompounds according to claim 1 or 2, characterised in that B is prolineor hydroxyproline.
 4. Prodrug compounds according to one of thepreceding claims, characterised in that C is an aminoacylpyrrolidide,aminoacylthiazolidide or N-dipeptidyl, O-acyl hydroxylamine.
 5. Prodrugcompounds according to one of the preceding claims, characterised inthat the inhibitors are present in salt form.
 6. Prodrug compoundsaccording to one of the preceding claims, characterised in that A-B is adipeptide of formula Ile-Pro or Gly-Pro.
 7. Pharmaceutical composition,especially for oral administration, characterised in that it comprisesat least one prodrug compound according to one of the preceding claimsoptionally in combination with customary carriers or excipients.
 8. Useof prodrug compounds or pharmaceutical compositions according to one ofthe preceding claims in the preparation of a medicament for thetemporally controlled in vivo inhibition of DP IV.
 9. Use of prodrugcompounds or pharmaceutical compositions according to one of claims 1 to6 in cell-, tissue- or organ-specific inhibition of DP IV.
 10. Use ofprodrug compounds or pharmaceutical compositions according to one ofclaims 1 to 6 in the treatment of disorders in mammals that can betreated by modulating the DP IV activity of a mammal.
 11. Use accordingto claim 9 in the treatment of metabolic disorders in humans.
 12. Useaccording to claim 9 in the treatment of impaired glucose tolerance,glucosuria, hyperlipidaemia, metabolic acidoses, diabetes mellitus,diabetic neuropathy and nephropathy and of sequelae of diabetes mellitusin mammals.